Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : III. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids

Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-¹⁴C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia.     

Purification and properties of three kinds of α-hydroxysteriod dehydrogenases from Brevibacterium fuscum DC33

The cell-free extract of Brevibacterium fuscum DC33 contained three kinds of hydroxysteriod dehydrogenase (3a-, 7a-, and 12a-hydroxysteriod dehydrogenases). 7a-Hydroxysteroid dehydrogenase (EC 1.1.1.59) was purified to electrophoretical homogeneity by ion exchange chromatography, affinity chromatography, and preparative electrophoresis. Its molecular weight was 104, 000 and the enzyme was composed of four identical subunits. The enzyme had an optimum pH of 5.3 for dehydrocholic acid reduction, and around 10 for cholic acid oxidation. It was stable in a pH range of 5.7 to 10.5 at 5°C overnight. The enzyme was most active at 25° to 30°C. The activity was not affected by incubation at 30°C for 30 min, but it was lost at 40°C for 30 min. Withe the assumption of two-substrate kinetics, we calculated various kinetic constants for dehydrocholic acid, 7, 12-diketolithocholic acid, 12-ketochenodeoxycholic acid, and 3, 12-diketolithocholic acid (for the structure of bile acids, see Table 2) together with NAD⁺ or NADH. The enzyme was active only toward hydroxysteroids with a 7a-hydroxyl group. The production of 7-ketochenodeoxycholic acid from cholic acid and of 3, 12-diketolithocholic acid from dehydrocholic acid by the purified 7a-hydroxysteroid dehydrogenase was confirmed by thin-layer chromatography.12a-Hydroxysteroid dehydrogenase was purified by a similar method. It was active toward hydroxysteroids with a 12a-hydroxyl group.3a-Hydroxysteroid dehydrogenase was purified by preparative electrophoresis. It was active toward hydroxysteroids with a 3a-hydroxyl group.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Paraffin Oxidation in Pseudomonas aeruginosa II. Gross Fractionation of the Enzyme System into Soluble and Particulate Components

Osmoplast production in Pseudomonas aeruginosa was investigated to obtain osmotically sensitive cells for studies on the subcellular location of the paraffin-oxidizing enzyme system. It proved possible to convert cells of P. aeruginosa treated with lysozyme and ethylenediaminetetraacetic acid in tris(hydroxymethyl)aminomethane-sucrose buffer (pH 8) into osmotically sensitive cells within 2 min. Active, cell-free preparations were obtained by the subsequent osmotic disruption in the presence of deoxyribonuclease and magnesium chloride. The conditions necessary for a complete separation of membranes and soluble cell constituents were established by following the distribution of two reference enzymes. An enzyme assay based on direct gas chromatographic analysis of the oxidation products from n-heptane is described for the paraffin-oxidizing enzyme system. By using this method, we investigated the enzymatic organization and subcellular distribution of the paraffin-oxidizing enzyme system. It was confirmed that the enzyme system is composed of three components, each of which is indispensable for the hydroxylation of n-heptane. One of these components, the hydroxylase, was located in two cell fractions; the other two components occur exclusively in the soluble cell fraction. The half-life of a crude enzyme preparation kept at ambient temperature is approximately 3.5 hr. This poor stability was found to be primarily due to the instability of one of the soluble factors, presumably the reduced nicotinamide adenine dinucleotide-rubredoxin reductase.     

Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis

Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca²⁺ or Mg²⁺ at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 °C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(ε-caprolactone), and poly(lactic acid).     

A developmental study of acid phosphatase-1 in Drosophila melanogaster.

The developmental profile of acid phosphatase-1 activity in Drosophila melanogaster indicates that this lysosomal gene-enzyme system (Acph-1, 3–101.1) is responsible for ca. 90% of the low-pH nucleotidase activity throughout development. The enzyme is present at particularly high levels during embryogenesis. It is shown with electrophoretic variants and null mutants of acid phosphatase-1 that virtually all of the embryonic enzyme is maternal in origin and is made during oogenesis. The enzyme exists in several isozymic forms at fertilization, and all but one of these forms disappear during early embryogenesis. Detectable maternal enzyme persists until the third larval instar stage. Crosses between females homozygous for a null allele and wild-type males show the zygotic Acph-1 gene activation occurs by at least 9 hr after oviposition.     

Geranylpyrophosphate: p-hydroxybenzoate geranyltransferase activity in extracts of Lithospermum erythrorhizon cell cultures

The enzymatic formation of m-geranyl-p-hydroxybenzoic acid from geranylpyrophosphate and p-hydroxybenzoic acid was investigated in cell-free extracts of Lithospermum erythrorhizon cell cultures. The reaction required the presence of a divalent cation, magnesium being the most effective activator. The enzyme showed a very broad pH optimum between pH 7.1 and 9.3. It was highly specific for both p-hydroxybenzoic acid and geranylpyrophosphate, and the apparent K_m values for these two substrates were 0.014 and 0.56 mM, respectively. The activity was located in the pellet of a 100 000 g centrifugation, showing that the enzyme is bound to membranes or microsomes. Shikonin-producing cultures contained an activity of this enzyme 35 times higher than non-producing cultures, suggesting that this enzyme is of regulatory importance in shikonin biosynthesis.     

Purification and Characterization of Gallic Acid Decarboxylase from Pantoea agglomerans T71

Oxygen-sensitive gallic acid decarboxylase from Pantoea (formerly Enterobacter) agglomerans T71 was purified from a cell extract after stabilization by reducing agents. This enzyme has a molecular mass of approximately 320 kDa and consists of six identical subunits. It is highly specific for gallic acid. Gallic acid decarboxylase is unique among similar decarboxylases in that it requires iron as a cofactor, as shown by plasma emission spectroscopy (which revealed an iron content of 0.8 mol per mol of enzyme subunit), spectrophotometric analysis (absorption shoulders at 398 and 472 nm), and inhibition of the enzyme activity by 2,2′-bipyridyl, o-phenanthroline, and EDTA. Another interesting feature of this strain is the fact that it contains a tannase, which is used together with the gallic acid decarboxylase in a two-enzyme resting cell bioconversion to synthesize valuable pyrogallol from readily available tannic acid.     

Purification and Characterization of a New Indole Oxygenase from the Leaves of Tecoma stans L

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to homogenity. The purified enzyme system catalyzes the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and 30°C. Two moles of oxygen are consumed and one mole of anthranilic acid is formed for every mole of indole oxidized. Dialysis resulted in complete loss of the activity. The inactive enzyme could be reactivated by the addition of concentrated dialysate. The enzyme is not inhibited by copper-specific chelators, non-heme iron chelators or atebrin. It is not a cuproflavoprotein, unlike the other indole oxygenases and oxidases.     

Virtual screening of mandelate racemase mutants with enhanced activity based on binding energy in the transition state

Mandelate racemase (MR) is a promising candidate for the dynamic kinetic resolution of racemates. However, the poor activity of MR towards most of its non-natural substrates limits its widespread application. In this work, a virtual screening method based on the binding energy in the transition state was established to assist in the screening of MR mutants with enhanced catalytic efficiency. Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening. The number of mutants for experimental validation was brought down to 17 by the virtual screening method, among which 14 variants turned out to possess improved catalytic efficiency. The variant V26I/Y54V showed 5.2-fold higher catalytic efficiency (k_cat/K_m) towards R-3-chloromandelic acid than that observed for the wild-type enzyme. Using this strategy, mutants were successfully obtained for two other substrates, R-mandelamide and R-2-naphthylglycolate (V26I and V29L, respectively), both with a 2-fold improvement in catalytic efficiency. These results demonstrated that this method could effectively predict the trend of mutational effects on catalysis. Analysis from the energetic and structural assays indicated that the enhanced interactions between the active sites and the substrate in the transition state led to improved catalytic efficiency. It was concluded that this virtual screening method based on the binding energy in the transition state was beneficial in enzyme rational redesign and helped to better understand the catalytic properties of the enzyme.     

A rapid and simple spectrophotometric assay of angiotensin-converting enzyme.

A rapid, simple, and accurate method for the chemical assay of anglotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensin-converting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a colorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-l-histidyl-l-leucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mm, and at HHL concentration of 3 mm, when the 5000g supernatant fluid of the rat lung was used.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum)

A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V_max per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M_r of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38°C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K_m values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V_max/K_m values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.     

A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

A simple radioactive assay system is elaborated for the measurement of dihyrofolate synthetase activity in Escherichia coli. It is also applicable to Neisseria gonorrhoeae and N. meningitidis extracts. Eight oxidized and reduced pteroate analogs have been examined for inhibitory activity. The most active inhibitor was dihydrohomopteroic acid followed by dihydro-10-thiopteroic acid, dihydrofolic acid, and dihydroisopteroic acid. The enzyme appears to be incapable of binding with substrate and any of the inhibitors in their oxidized forms.     

A fluorescent assay for bacterial cell wall lytic enzymes

A sensitive fluorimetric assay is described for the measurement of N-acetylmuramic acid l-alanine amidase as well as lysozyme. The method uses Bacillus subtilis cell walls labeled with fluorescamine on the free amino group of diaminopimelic acid. The method can easily detect the lytic activity of 0.02 μg of pure N-acetylmuramic acid l-alanine amidase in 30 min and of 1 μg of hen egg-white lysozyme in the same period. The method is particularly suitable for measurement of competition between various cell wall preparations for the same enzyme.     

Determination of l-galactose in polysaccharide material

A method is described for the identification and quantitative determination Of l-galactose in hydrolyzates of polysaccharide material. In this technique, all the d-galactose is oxidized to d-galactonic acid using the enzyme d-galactose dehydrogenase. Remaining sugars, including any l-galactose, are converted to their trimethylsilyl derivatives and estimated by GLC. l-Galactose was detected in polysaccharides of flax seed, corn cob and corn root, and in the cell wall of Acer pseudoplatanus suspension cultures. It is suggested that the sugar may be relatively widespread in plants.     

Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile

A nitrilase that converts racemic mandelonitrile to R-(—)-mandelic acid was purified to apparent homogeneity from a cell extract of Alcaligenes faecalis ATCC 8750. The molecular weight of this enzyme was estimated to be 32,000±2,000 from SDS-PAGE and that of the native enzyme 460,000±30,000 from HPLC gel filtration. The enzyme preferentially hydrolyzed substituted aliphatic nitriles, in particular benzyl cyanide and its p-substituted compounds, but hydrolyzed aromatic nitriles only with difficulty. The amino-terminal amino acids were sequenced and their sequences compared with those of other nitrilases. The purified enzyme had a pH optimum of 7.5 and an optimum temperature range of 40 to 45°C. The enzyme was inhibited by various thiol reagents. It hydrolyzed racemic mandelonitrile, producing optically pure R-(—)-mandelic acid and ammonia without the concomitant production of mandelamide, evidence that this nitrilase is highly enantioselective for R-mandelonitrile.     

Effector controlling accumulation of N-acetylglucosaminidase during development of Dictyostelium discoideum

The accumulation of N-acetylglucosaminidase, an early developmentally regulated enzyme in Dictyostelium discoideum, is dependent upon the action of a heat-stable effector molecule secreted by the cells. Stimulation of enzyme accumulation is inhibited by cycloheximide and actinomycin, suggesting that it requires concomitant RNA and protein synthesis. The effector elutes from Sephadex columns as a molecule of 300 to 1000 daltons. It is stable to treatment with a variety of proteolytic enzymes and mild acid hydrolysis but can be inactivated by prolonged acid hydrolysis.     

Expression chez Escherichia coli de la décarboxylase des acides aminés aromatiques de rat sous forme d'une protéine de fusion active

The DNA sequence encoding rat aromatic-L-amino acid decarboxylase (AADC) was inserted into the Escherichia coli (E. coli) expression vector pMAL-c2. This clone produced a fusion protein able to catalyze the conversion of L-DOPA to dopamine. After purification and treatment of the fusion protein by factor Xa (FXa), an enzymatically active form of the enzyme resistant to FXa was isolated. It showed kinetic constants, V_max, K_m, and enzymatic properties very similar to those obtained previously for the mammalian enzyme. This method for obtaining active AADC appears to be useful for initiating the study of the catalytic activity of this protein because it permitted the rapid isolation and the stabilization of an active form of the enzyme.     

Purifications and properties of L-mandelate- 4-hydroxylase from Pseudomonas convexa.

An inducible l-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe²⁺, and O₂ for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with l-mandelate, NADPH, and ferrous sulfate and K_m values for these substrates were found to be 1 × 10^?4, 1.9 × 10^?4, and 4.7 × 10^?5m, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.